Serum and cerebrospinal fluid neurofilament light chain and glial fibrillary acid protein levels in early and advanced stages of cerebral amyloid Angiopathy

Background Neurofilament light chain (NFL) is a biomarker for neuroaxonal damage and glial fibrillary acidic protein (GFAP) for reactive astrocytosis. Both processes occur in cerebral amyloid angiopathy (CAA), but studies investigating the potential of NFL and GFAP as markers for CAA are lacking. We aimed to investigate NFL and GFAP as biomarkers for neuroaxonal damage and astrocytosis in CAA. Methods For this cross-sectional study serum and cerebrospinal fluid (CSF) samples were collected between 2010 and 2020 from controls, (pre)symptomatic Dutch-type hereditary (D-CAA) mutation-carriers and participants with sporadic CAA (sCAA) from two prospective CAA studies at two University hospitals in the Netherlands. NFL and GFAP levels were measured with Simoa-assays. The association between NFL and GFAP levels and age, cognitive performance (MoCA), CAA-related MRI markers (CAA-CSVD-burden) and Aβ40 and Aβ42 levels in CSF were assessed with linear regression adjusted for confounders. The control group was divided in age < 55 and ≥55 years to match the specific groups. Results We included 187 participants: 28 presymptomatic D-CAA mutation-carriers (mean age 40 years), 29 symptomatic D-CAA participants (mean age 58 years), 59 sCAA participants (mean age 72 years), 33 controls < 55 years (mean age 42 years) and 38 controls ≥ 55 years (mean age 65 years). In presymptomatic D-CAA, only GFAP in CSF (7.7*103pg/mL vs. 4.4*103pg/mL in controls; P<.001) was increased compared to controls. In symptomatic D-CAA, both serum (NFL:26.2pg/mL vs. 12.5pg/mL; P=0.008, GFAP:130.8pg/mL vs. 123.4pg/mL; P=0.027) and CSF (NFL:16.8*102pg/mL vs. 7.8*102pg/mL; P=0.01 and GFAP:11.4*103pg/mL vs. 7.5*103pg/mL; P<.001) levels were higher than in controls and serum levels (NFL:26.2pg/mL vs. 6.7pg/mL; P=0.05 and GFAP:130.8pg/mL vs. 66.0pg/mL; P=0.004) were higher than in pre-symptomatic D-CAA. In sCAA, only NFL levels were increased compared to controls in both serum (25.6pg/mL vs. 12.5pg/mL; P=0.005) and CSF (20.0*102pg/mL vs 7.8*102pg/mL; P=0.008). All levels correlated with age. Serum NFL correlated with MoCA (P=0.008) and CAA-CSVD score (P<.001). NFL and GFAP in CSF correlated with Aβ42 levels (P=0.01/0.02). Conclusions GFAP level in CSF is an early biomarker for CAA and is increased years before symptom onset. NFL and GFAP levels in serum and CSF are biomarkers for advanced CAA. Supplementary Information The online version contains supplementary material available at 10.1186/s13195-024-01457-0.


Serum NFL and GFAP measurements
At baseline, blood was withdrawn via standard vena puncture and stored at room temperature for a maximum of 2 hours until centrifuging (2350 relative centrifugal force (RCF) for 10 minutes at 20° C) and storage in aliquots in polypropylene tubes at -80°C.The commercially available Simoa™ NF-Light Advantage Kit (Quanterix) and Simoa™ GFAP Discovery Kit (Quanterix) were used according to manufacturer's instructions except for GFAP serum analysis where a 8 fold sample dilution was applied due to sample volume restrictions, as previously described. 1The serum samples were randomized and centrifuged prior to use (3 min; 14000g) followed by manual 4 fold respectively 8 fold dilution for NFL and GFAP into a 96 well microplate.Next, sample testing was done in duplicate over two consecutive test runs.For the GFAP analysis the samples received an additional freezethaw step compared to the NFL analysis.Samples with %CV>20 were excluded from the analysis.An average intra-assay %CV of 3.4% (range 0.0-17.8)for NFL and 5.8% (range 0.0 -19.8) for GFAP were obtained, samples with an intra-assay CV >15% were re-run for NFL and then excluded from further analyses when intra-assay was >20%.

CSF NFL and GFAP measurements
We collected CSF samples of all participants who consented to undergo a lumbar puncture, all samples were obtained under standardized conditions at the same time frame.CSF was collected in polypropylene tubes and transferred to the laboratories within 30 minutes.We centrifuged (2350 RCF for 10 minutes at 20°C) the samples and stored them in aliquots in polypropylene tubes at -80°C.
CSF NFL and GFAP levels were quantified using Simoa Kits, as mentioned above according to the manufacturer's instructions.The CSF samples were randomized and centrifuged prior to use (3 min; 14000g) followed by manual 100 fold respectively 40 fold dilution for NFL and GFAP into a 96 well microplate.Next, sample testing was done in duplicate in a single test runs.

MRI protocol
Leiden: MRI was performed on a 3.0 Tesla MRI scanner (Philips Achieva, Best, the Netherlands) in all D-CAA and sCAA participants and data was acquired using a standard 32-channel head coil.Three- Tesla MRI scan (Siemens Magneto, Siemens Healthcare,Erlangen, Germany) with a 32-channel head coil.Participants were examined using a comprehensive protocol, and the for the current study, the 3D multi-echo gradient echo T2*-weighted sequence (voxel size 0.8 x 0.8 x0.8 mm), the 3D T2weighted sequence (voxel size 0.8 x 0.8 x 0.8 mm) and 3D fluid-attenuated inversion recovery (FLAIR) sequence (voxel size 0.8 x 0.8 x0.8 mm) were analyzed.Magnitude and phase data from the multi-echo gradient sequence was processed to a SWI using the "Contrast-weighted, Laplaceunwrapped, bipolar multi-Echo, ASPIRE-combined, homogeneous, improved Resolution SWI" (CLEAR-SWI) method. 2

Additional information on MRI assessment
Macrobleeds and microbleeds were scored on SWI images.cSS was scored on SWI images, the focality score was obtained according to previously published classifications. 3,4 erivascular spaces (PVS) were assessed on T2-weighted MRI and Fluid-attenuated inversion recovery (FLAIR) images were used to assess white matter hyperintensities (WMH).
T1 weighted images were acquired with the following parameters: repetition time(TR)/ echo time (TE) = 9.7/4.6ms,flip angle 7 degrees, 130 slices with no interslice gap and a field of view (FOV) of 217 x 172 x 156 mm with a voxel size of 1.2 x 1.2 x 1.2 mm, resulting in a scan duration of 2:48 min.T2 weighted images were acquired with the following parameters: TR/ TE = 4744/80ms, flip angle 90 degrees, 48 slices with no interslice gap and an FOV of 220 x 176 x 144 mm with a voxel size of 0.5 x 0.6 x 3 mm, resulting in a scan duration of 2:13 min.Three dimensional Fluid Attenuated Inversion Recovery (FLAIR) images were acquired with the following parameters: TR/TE = 4800/280ms, inversion time (TI) 1650ms , 321 slices with no interslice gap and an FOV of 250 x 250 x 180 mm with a voxel size of 1.1 x 1.1 x 0.6 mm, resulting in a scan duration of 4:43 min.Susceptibility weighted images (SWI) were acquired using the following parameters: TR/TE = 31/7.2ms,flip angle 17 degrees, 130 slices and an FOV of 230 x 190 x 130 mm with a voxel size of 0.6 x 0.6 x 1 mm resulting in a scan duration of 3:31 min.Nijmegen:The CAA participants from the CAVIA study underwent a 1.5 or 3.0 Tesla MRI of the brain using different MRI systems with varying protocols in the RUMC or referral hospitals, which at least included T2*-weighted images or susceptibility-weighted imaging sequence (SWI) images, fluidattenuated inversion recovery (FLAIR) and T2 sequences.The BIONIC participants underwent a 3.0